CN108977381A - Raw bacillus amyloliquefaciens and its application in a kind of banana - Google Patents
Raw bacillus amyloliquefaciens and its application in a kind of banana Download PDFInfo
- Publication number
- CN108977381A CN108977381A CN201810888397.0A CN201810888397A CN108977381A CN 108977381 A CN108977381 A CN 108977381A CN 201810888397 A CN201810888397 A CN 201810888397A CN 108977381 A CN108977381 A CN 108977381A
- Authority
- CN
- China
- Prior art keywords
- bacillus amyloliquefaciens
- banana
- bacteria
- bacterium
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides raw bacillus amyloliquefaciens and its application in a kind of banana, the bacterium be named as bacillus amyloliquefaciens (Bacillus amyloliquefaciens) G9R-3, CCTCC NO:M 2017747；Preservation place are as follows: China, Wuhan, Wuhan University；China typical culture collection center, preservation time are on November 30th, 2017.The invention has the following advantages: one plant of new bacillus amyloliquefaciens is isolated in screening, which has the inhibiting effect of excellent to banana blight bacteria, has important practical significance for prevention and treatment banana blight.
Description
Technical field
The invention belongs to bacillus amyloliquefaciens raw in field of biotechnology more particularly to a kind of banana and its applications.
Background technique
Banana blight also known as Panama disease and yellowtop are by Fusarium oxysporum Cuba specialized form (Fusarium
Oxysporum f.sp.cubense, abbreviation Foc) caused by a kind of vascular bundle necrosis crushing banana fungal disease and typical case
Soil-borne disease.The disease route of transmission is wide, and pathogenicity is strong, and rate of propagation is fast, and infection rate is high, and prevention and treatment is difficult, dead after plant is infected
Rate height is died, referred to as " banana cancer ".Banana blight is very tight to the harm of banana industry Yi Dan generation with regard to great destructiveness
Weight.This disease is found for 1874 in Australia, and the middle of last century is broken out in several national large area of Panama and South America,
There are about 43hm2Banana garden ruined, so that worldwide international trade banana variety rice seven (Gros Michel) is exited history
Stage, and the whole world is traveled to by banana export.To the fifties in last century, in addition to the South Pacific Ocean, Mediterranean, Malay
Outside some island, there is the generation of banana blight in the world elsewhere.
Endophyte of plant refer to those in its history of life a certain stage life within plant tissue, not to plant tissue
The microorganism for causing obvious Disease symptoms, the saprophytic microbe of battalion of a certain stage surface life including those in its history of life
With the latent disease pathogenic microorganism and mycorrhizal fungi not injured temporarily to host.The research of endophyte of plant starts from 1997
The research to Festuca Arundinacea endogenetic fungus such as Bacon, subsequent people to numerous crops, as beet, tobacco, Radix Glycyrrhizae, capsicum, citrus,
The endogenetic bacterias such as cotton, rice, "Hami" melon have carried out research.It is closed research shows that many endophytes and host form mutually beneficial symbiosis
System, i.e. host provide sufficient nutrition for endophyte and promote its growth.Simultaneously endophyte host cope with external environment stress
Tolerance (including drought resisting, disease and insect resistance and to pathogen antagonism etc.), the growth of host plant, the effective active in host plant
Generation of ingredient etc. also has an important influence on.The mechanisms of control wheat scab of endophyte of plant is mainly manifested in by generating antibiotic
Class, hydrolase, plant growth regulator and alkaloids substance compete nutriment with pathogen, enhance host plant
The approach such as resistance and induction plant generation system resistance inhibit growth of pathogenic bacteria.Research also shows to be inoculated with healthy banana plant
The tissue culture seedlings of bananas of middle endophyte can not only mitigate the disease incidence of wilt disease, can also promote the growth of plant.
In plant disease biocontrol bacteria, using it is more be bacillus (Bacillus spp.).It is a kind of important
Microbial resources can be resistant to because it can generate the gemma of heat-resisting, drought-enduring, uvioresistant, electromagnetic radiation and organic solvent
Various undesirable environmental conditions.Field information it has proven convenient that the biocontrol agent of bacillus in stability, compatible with chemical pesticide
Property and different plant different year preventive effects in terms of be substantially better than non-bacillus and fungi biocontrol agent.And
Bacillus can be made into pulvis, liquor.Pulvis be easy storage, be readily transported, large-scale production process is simple, cost compared with
It is low.Therefore, bacillus is a kind of current ideal Biocontrol microorganism.
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of bacillus amyloliquefaciens, which is named as solution starch bud
Spore bacillus (Bacilus amyloliquefaciens) G9R-3, CCTCC NO:M 2017747；Preservation place are as follows: China, it is military
The Chinese, Wuhan University；China typical culture collection center, preservation time are on November 30th, 2017.
In the present invention, it further illustrates, the described bacterial strain well-grown, bacterium colony on LA culture medium or NA culture medium are in
Canescence, edge is irregular, opaque, adheres, extension.Microscopy is in rod-short, has flagellum, there is gemma；PH=4.0-9.0,
It can be grown in the environment of 20 DEG C -50 DEG C；Gram's staining is positive；Oxidizing ferment, catalase are measured as the positive；Methyl red
(M.R) measurement is negative；In pH=7.0, V-P measurement generates red compound；The detection of 3- ketone group lactose is negative；Aesculin
Hydrolyze tests positive；Cellulose hydrolysis, Starch Hydrolysis detection are negative；Milk hydrolysis, glycitols fermentation tests positive；It is bright
Dispergation test is positive；Urase reaction is positive；Esterase reaction, arginine dihydrolase detection are negative；Containing quality
It can be grown on the LA culture medium and NA culture medium of the NaCl of concentration 2%-5%.
Molecular Identification
The 16S rDNA complete sequence of bacillus amyloliquefaciens G9R-3CCTCC No:M 2017747 is as shown in sequence 1, overall length
For 1427bp.
Correspondingly, the present invention also provides a kind of separating screening method of bacillus amyloliquefaciens raw in banana, including it is following
Several steps:
A: the root tissue after taking banana surface sterilization is added sterile water and is ground, take filtrate, dilute in mortar
Afterwards, it takes dilution to be coated on NA culture medium flat plate, after culture, draws the sterile water spread plate inspection of last time cleaning sample
Whether thorough survey disinfection；All single colonies of picking transfer scribing line on new NA culture medium flat plate respectively, isolate and purify；It will purifying
Endogenetic bacteria single colonie afterwards is inoculated in the bottle of the culture solution containing NA, shakes bacterium, is mixed, is saved backup with the glycerol of sterilizing；
B: the bacterium for having bacteriostasis to banana blight bacteria is filtered out from the bacterium colony of step A picking with tablet face-off method
Strain.
Preferably, in the step A, disinfection uses mass concentration to rinse 1-2min, sterile water wash 1 for 75% alcohol
Secondary, being put into mass concentration is that 15-20min is impregnated in 3.25% active sodium hypochlorite, in the ethanol solution that mass concentration is 75%
Then surface sterilization 1min is used sterile water wash 3-5 times.
Preferably, in stepb, the tablet face-off method specific steps are as follows: cause the banana blight that diameter is 10mm
The bacteria cake of germ FOC4 is inoculated in PDA culture medium plate center, and the bacterium colony separated with the oese picking of sterilizing is away from bacteria cake two
It crosses, is inverted at the 2.5cm of side, cultivated at 26 DEG C -28 DEG C, observe bacterial strain fungistatic effect.
Preferably, in the step A, endogenetic bacteria single bacterium after purification is mixed with the glycerol of sterilizing with 1:1.Wherein, sweet
The mass concentration of oil is 30%-40%.
The present invention also provides application of the bacillus amyloliquefaciens in banana blight bacteria prevention and treatment.
The invention has the following advantages: one plant of new bacillus amyloliquefaciens is isolated in screening, the bacterial strain is to banana
Wilt has the inhibiting effect of excellent, has important practical significance for prevention and treatment banana blight.
Detailed description of the invention
Fig. 1 is bacterial strain flat board photo of the present invention.
Fig. 2 is inhibiting effect of the lipopeptide compound crude extract to Foc4 of endogenetic bacteria G9R-3 various concentration of the present invention.
Fig. 3 is inhibiting effect of the bacterial strain flat board to Foc4.
Fig. 4 is inhibiting effect of the escaping gas to Foc4.
Fig. 5 is fungicide potting cup seedling control effect.
Specific embodiment
With reference to the accompanying drawing, preferably embodiment of the invention is described in further detail:
Lipopeptid class functional gene with synthesis antagonism
Discovery solution starch bud is detected by polymerase chain reaction (Polymerase Chain Reaction, PCR) method
Spore bacillus G9R-3 has the lipopeptid class functional gene of synthesis antagonism, extracts bacillus amyloliquefaciens G9R-3DNA, referring to text
The primer for offering report, using bacterial strain DNA as template amplification lipopeptid class functional gene, discovery G9R-3 contains lipopeptide antibiotic
The biosynthesis related genes of fenA, ituC, srfA.
Method preparation in the primer reference literature: Cheng Kai prevention and treatment soil passes the development of cotton verticillium wilt microbial organic fertilizer
With the Nanjing biological effect research [D]: Agricultural University Of Nanjing, 2010.
Gene with synthesis 1,4 beta-glucanase
It is detected by polymerase chain reaction (Polymerase Chain Reaction, PCR) method, to solve starch bud
Spore bacillus G9R-3DNA is template, and the primer of reference literature report expands the gene for obtaining synthesis 1,4 beta-glucanase, and the gene is complete
A length of 685bp.
Embodiment 1
The separating screening method of raw bacillus amyloliquefaciens in a kind of banana, the screening specifically includes the following steps:
A: the root tissue after taking banana surface sterilization is added appropriate amounts of sterilized water and is ground, take filtrate in mortar, dilute
It releases to after 10-1000 times, 100 μ L of dilution is taken to be coated on NA culture medium flat plate, after 28 DEG C of culture 72h, draw last time
Whether the sterile water 0.2mL spread plate detection disinfection of cleaning sample is thorough.All single colonies of picking transfer scribing line new respectively
NA culture medium flat plate on, isolate and purify.Endogenetic bacteria single colonie after purification is inoculated in the triangle of the culture solution of NA containing 20mL
In bottle, 120rpm shakes bacterium for 24 hours, is mixed with 40% glycerol of sterilizing with 1:1, -80 DEG C save backup.
B: the bacterium for having bacteriostasis to banana blight bacteria is filtered out from the bacterium colony of step A picking with tablet face-off method
Strain, as shown in Figure 1, being bacterial strain flat board photo.
In the present invention, it further illustrates, in step, the concrete operations of disinfection are the wine for being 75% with mass concentration
Essence rinsing 2min, sterile water wash 1 time, is put into 3.25% active sodium hypochlorite and impregnates 15min, surface in 75% ethanol solution
1min is sterilized, is then used sterile water wash 3-5 times；
In stepb, the tablet face-off method specific steps are as follows: the banana blight pathogenic bacteria FOC4 for being 10mm by diameter
Bacteria cake, be inoculated in PDA culture medium plate center, the bacterium colony separated with the oese picking of sterilizing is away from bacteria cake two sides 2.5cm
Place's scribing line, is inverted, and cultivates at 28 DEG C, observes bacterial strain fungistatic effect.
Endogeny measurement
The bacterial strain G9R-3-Rif that anti-rifampin label is obtained using the method for induction of resistance, is inoculated with after Banana Seedlings respectively
Bacterium colony recycling is carried out on plate referring to above-mentioned separating screening method in the 3rd day, 5 days.
The method that fungicide is prepared by bacillus amyloliquefaciens G9R-3CCTCC NO:M 2017747
The bacillus amyloliquefaciens G9R-3 for taking inclined-plane NA culture medium to save, scribing line to LB culture medium flat plate, is placed in 37 DEG C,
8h is cultivated, after growing single colonie, the triangular flask (50mL) of dress 20mL LB culture solution is seeded to sterile toothpick picking G9R-3
It is interior, it is placed on shaking table, under the conditions of 37 DEG C, with 160rpm/min constant-temperature shaking culture 2-3h；Then aseptically according to
The inoculum concentration access that volumn concentration is 1% is equipped in the triangular flask (500mL) of 200mL LB culture solution；Shaking flask after inoculation
Under the conditions of 37 DEG C, the speed oscillation culture 14h of 160rpm/min obtains bacillus amyloliquefaciens G9R-3 fermentation liquid, that is, sterilizes
Agent.Fig. 2 is that the lipopeptide compound crude extract of endogenetic bacteria G9R-3 various concentration of the present invention is to the inhibiting effect of Foc4, Fig. 3
Bacterial strain flat board is inhibiting effect of the escaping gas to Foc4 to the inhibiting effect of Foc4, Fig. 4.
The fungicide of the present embodiment contains bacillus amyloliquefaciens G9R-3 thallus and metabolite, individual thallus and generation
Thanking to product has bacteriostasis.
Embodiment 2
The fungicide of preparation is to banana blight bacteria (Fusarium oxysporum f.sp.cubense) strain growth
Inhibiting effect
After plate activates, 28 DEG C of culture 3d on PDA plate are beaten with punch in colony edge Fusarium oxysporum Foc4
Take the bacteria cake of diameter 5mm spare.It is separately added into fungicide filtrate into the 50m L PDA after dissolving, is poured into culture dish
The malicious plate of band is made in (90mm), is inoculated with Foc4 bacteria cake after cooling plate, Foc4 bacteria cake is inoculated with as blank using nontoxic PDA plate
Control is repeated 3 times using only adding anhydrous methanol as solvent control.Pathogen strain growth situation is observed after 7d, is handed over using cross
Fork method measures Foc4 colony diameter and calculates bacteriostasis rate: bacteriostasis rate (%)=(control colony diameter processing bacterium according to following formula
Fall diameter)/(control colony diameter-bacteria cake diameter) × 100.The result shows that fungicide is to blight banana wilt
The inhibiting rate of (Fusarium oxysporum f.sp.cubense) strain growth reaches 65.92 ± 2.31%.
Embodiment 3
Lipopeptid substance crude extract in microbial inoculum is to banana blight bacteria (Fusarium oxysporum
F.sp.cubense) the inhibiting effect of mycelia
The XAD-16 macroreticular resin that 10m L is handled well is added to fungicide supernatant, was slowly stirred on magnetic stirring apparatus
Night, filter paper filter liquid.Resin is eluted after pure water resin 2 times, then with anhydrous methanol.The eluent vacuum of collection
10mgm L is made with methanol solution dissolution after weighing in freeze drier freeze-drying-1Crude extract mother liquor.To the 50m after dissolving
Crude extract after being separately added into gradient dilution in L PDA simultaneously makes its final concentration be respectively 10,50,100,200,300 μ gm L-1,
It is poured into culture dish (90mm) and the malicious plate of band, after cultivating Foc4 bacterial strain 7d, picking colony edge mycelia, without place is made
The normal mycelia of reason is blank control, and the mycelia handled using solvent anhydrous methanol is solvent control, 0.04% Trypan Blue
After 10min, micro- sem observation hypha form and staining conditions are used.As the final concentration of 10 μ gmL of crude extract-1When to Foc4's
Mycelia growth begins with inhibiting effect, and works as the final concentration of 300 μ gm L of crude extract-1When can almost completely inhibit Foc4 mycelia
Growth, inhibiting rate is up to 56.91 ± 3.24%；Solvent methanol to Foc4 without significantly inhibit effect (P > 0.05).
Embodiment 4
Fungicide volatile materials is raw to banana blight bacteria (Fusarium oxysporum f.sp.cubense) bacterial strain
Long inhibiting effect
By Antagonistic Endophytic, 37 DEG C of shake cultures are for 24 hours in LB liquid medium.Each LB plate places 5 filter paper
20 μ L endogenetic bacteria bacterium solutions, 28 DEG C of stationary culture 7d after sealing is added in piece, each filter paper.
It is inoculated with pathogen bacteria cake in freshly prepd PDA plate center, is tipped upside down on and has been cultivated, with endogenetic bacteria
It on LB plate ware bottom, is sealed with Parafilm film, using directly pathogen plate simultaneously, as blank control, each processing is repeated 3 times.
28 DEG C co-culture, and the growing state of pathogen is observed after 7d, measure Foc4 colony diameter using crossing method and according to following
Formula calculates bacteriostasis rate: bacteriostasis rate (%)=(control colony diameter handles colony diameter)/(control colony diameter-bacteria cake diameter)
×100.Test result shows volatile materials to banana blight bacteria (Fusarium oxysporum f.sp.cubense)
The inhibiting rate of bacterial strain reaches 55.64 ± 0.90%.
The growth-promoting functions of 5 microbial inoculum tieback difference banana variety cup seedling of embodiment
It chooses 4 kinds of different banana germplasm (ten thousand clocks 4, osmanthus any of several broadleaf plants No. 9, osmanthus any of several broadleaf plants No. 1, extra large tribute any of several broadleaf plants) sand bed seedlings and is transferred to coco bran band soil
Cup seedling in, in addition to routinely fertilizer and water management, directly filled every 10d and apply G9R-3 bacterium solution, bacterium solution amount of application press cup seedling volume calculations
(concentration is 1 × 106Cfu/mL, 100mL/ glasss), application equivalent LB liquid medium is blank control.After application bacterium solution 3 times, point
It does not take different 200 plants of banana germplasm cup seedling of application endophyte liquid and LB liquid medium respectively, is measured after being inoculated with 30d with tape measure
Banana seedlings plant height (height of the false stem to new blade root)；The banana seedlings that weigh with scale fresh weight；And its number of blade is counted, such as
Shown in table 1.
The growth-promoting functions of 1 endogenetic bacteria G9R-3 tieback difference banana variety cup seedling of table measure
Note: different letters indicate difference up to 0.05 level of signifiance after numerical value.
Embodiment 5: microbial inoculum tieback difference banana variety cup seedling improves banana blight resistance
G9R-3 tieback difference banana variety (osmanthus any of several broadleaf plants No. 1, cloud grind No. 2, ten thousand clock 4, osmanthus any of several broadleaf plants No. 9) cup seedling-growing method is referring to real
Example 4 after last time applies G9R-3 bacterium solution 10d, respectively takes the banana variety cup seedling of 200 plants of different disposals, root-pouring method inoculation respectively
(concentration is 1 × 10 to Foc4 spore suspension6Cfu/mL, 100mL/ glasss), plant incidence is investigated after 30d.Count disease incidence
And disease index and relative control effect are calculated as follows referring to disease index grade scale.The result shows that (as shown in table 2), is killed
The use of microbial inoculum can significantly reduce different banana variety wilt disease disease incidence.
Disease incidence (%)=wilt disease morbidity effective strain × 100 of strain/test
Disease index=[Σ (the diseased plant numbers at different levels × disease grade)]/(investigating total strain tree × highest disease grade) × 100
Relative control effect (%)=(control disease index-processing disease index)/control disease index × 100
Grade scale refers to NY/T2248-2012. tropical crops variety source disease and insect resistance identification technology regulation-Banana Leaf
Pinta, banana blight and the Beijing banana root knot nematode disease [S]: China Standards Press, 2012.
The disease resistance evaluation of 2 endogenetic bacteria G9R-3 tieback difference banana variety cup seedling of table
Note: different letters indicate difference up to 0.05 level of signifiance after numerical value.
Also, as shown in figure 5, inoculation endogenetic bacteria G9R-3 processing preventive effect is not inoculated with No. 1 leaf of osmanthus any of several broadleaf plants of control up to 65%
Piece yellow, plant are wilted dead.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
Specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to of the invention
Protection scope.
Sequence table
<110>Guangxi Autonomous Region Academy of Agricultural Sciences's biotechnology research institute
<120>raw bacillus amyloliquefaciens and its application in a kind of banana
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1427
<212> DNA
<213>bacillus amyloliquefaciens ((Bacillus amyloliquefaciens))
<400> 1
aatacatgca agtcgagcgg acagatggga gcttgctccc tgatgttagc ggcggacggg 60
tgagtaacac gtgggtaacc tgcctgtaag actgggataa ctccgggaaa ccggggctaa 120
taccggatgg ttgtctgaac cgcatggttc agacataaaa ggtggcttcg gctaccactt 180
acagatggac ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca aggcgacgat 240
gcgtagccga cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct 300
acgggaggca gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc 360
gtgagtgatg aaggttttcg gatcgtaaag ctctgttgtt agggaagaac aagtgccgtt 420
caaatagggc ggcaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc 480
agccgcggta atacgtaggt ggcaagcgtt gtccggaatt attgggcgta aagggctcgc 540
aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg tcattggaaa 600
ctggggaact tgagtgcaga agaggagagt ggaattccac gtgtagcggt gaaatgcgta 660
gagatgtgga ggaacaccag tggcgaaggc gactctctgg tctgtaactg acgctgagga 720
gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga 780
gtgctaagtg ttagggggtt tccgcccctt agtgctgcag ctaacgcatt aagcactccg 840
cctggggagt acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagcg 900
gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatcctc 960
tgacaatcct agagatagga cgtccccttc gggggcagag tgacaggtgg tgcatggttg 1020
tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct 1080
tagttgccag cattcagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg 1140
tggggatgac gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg 1200
acagaacaaa gggcagcgaa accgcgaggt taagccaatc ccacaaatct gttctcagtt 1260
cggatcgcag tctgcaactc gactgcgtga agctggaatc gctagtaatc gcggatcagc 1320
atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt 1380
gtaacacccg aagtcggtga tagtaacctt tatggagcca gccgccg 1427
<210> 2
<211> 759
<212> DNA
<213>bacillus amyloliquefaciens ((Bacillus amyloliquefaciens))
<400> 2
taattccatg gatgttttat cgtatgaaac gagtgctgct gcttcttgtc actggattgt 60
ttttgagttt gtgtgcgatc acttctactg catcagctca aacaggcgga tcgttttttg 120
aaccttttaa cagctataac tccggattat ggcaaaaagc aaatggttat tccaatggag 180
atatgttcaa ctgcacgtgg cgtgcaaata atgtatcaat gacgtcatca ggtgaaatgc 240
gtttggcgct gacaagcccg tcttataaca agtttgactg cggggagaac cgctccgttc 300
aaacctatgg ctatggactt tatgaagtca gaatgaaacc ggctaaaaac acagggatcg 360
tttcatcgtt cttcacttat acaggtccaa cggatggcac tccttgggat gagattgata 420
tcgaattttt aggaaaagac acgacaaagg ttcaatttaa ctattataca aatggcgcgg 480
gaaaccatga gaaggttgcg gatctcggat ttgacgcggc caatgcctat catacgtatg 540
cgttcgattg gcagccaaac tctattaaat ggtatgtcga tgggcaatta aaacatactg 600
cgacaagcca aatcccgaca aacccgggaa agatcatgat gaacttgtgg aatgggatag 660
gtgtcgatga ctggctcggt tcctacaatg gtgtaaatcc gctatacgct cattatgact 720
gggtgcgcta tacaaaaaaa taaggatcca attaatctc 759
Claims (8)
1. a kind of bacillus amyloliquefaciens, which is characterized in that the bacterium be named as bacillus amyloliquefaciens (Bacillus amyloliquefaciens) G9R-3, CCTCC NO:M 2017747；Preservation place are as follows: China, Wuhan, Wuhan University；In
State's Type Tissue Collection, preservation time are on November 30th, 2017.
2. bacillus amyloliquefaciens as described in claim 1, which is characterized in that the bacillus amyloliquefaciens G9R-3
The 16S rDNA complete sequence overall length of CCTCC No:M 2017747 is 1427bp, as shown in SEQ ID NO1.
3. bacillus amyloliquefaciens as described in claim 1, which is characterized in that it expands the base for obtaining synthesis 1,4 beta-glucanase
Cause, the full length gene are 685bp, and sequence is as shown in SEQ ID NO2.
4. the separating screening method of raw bacillus amyloliquefaciens in a kind of banana, which is characterized in that including the following steps:
A: the root tissue after taking banana surface sterilization is added sterile water and is ground, take filtrate, after dilution, take in mortar
Dilution is coated on NA culture medium flat plate, and after culture, the sterile water spread plate detection for drawing last time cleaning sample disappears
Whether poison is thorough；All single colonies of picking transfer scribing line on new NA culture medium flat plate respectively, isolate and purify；It will after purification
Endogenetic bacteria single colonie is inoculated in the bottle of the culture solution containing NA, shakes bacterium, is mixed, is saved backup with the glycerol of sterilizing；
B: the bacterial strain for having bacteriostasis to banana blight bacteria is filtered out from the bacterium colony of step A picking with tablet face-off method.
5. method as claimed in claim 4, which is characterized in that in the step A, disinfection rinses 1-2 min, nothing using alcohol
The cleaning of bacterium water, is put into active sodium hypochlorite and impregnates, then sterile water wash is used in surface sterilization in ethanol solution.
6. method as claimed in claim 4, which is characterized in that in stepb, the tablet face-off method specific steps are as follows: will
The bacteria cake of banana blight pathogenic bacteria FOC4 is inoculated in PDA culture medium plate center, the bacterium separated with the oese picking of sterilizing
It falls in away from crossing at bacteria cake two sides, is inverted, bacterial strain fungistatic effect is observed in culture.
7. method as claimed in claim 4, which is characterized in that in the step A, endogenetic bacteria single bacterium and sterilizing after purification
Glycerol mixed with 1:1.
8. application of the bacillus amyloliquefaciens described in claim 1 in banana blight bacteria prevention and treatment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810888397.0A CN108977381A (en) | 2018-08-07 | 2018-08-07 | Raw bacillus amyloliquefaciens and its application in a kind of banana |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810888397.0A CN108977381A (en) | 2018-08-07 | 2018-08-07 | Raw bacillus amyloliquefaciens and its application in a kind of banana |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108977381A true CN108977381A (en) | 2018-12-11 |
Family
ID=64555983
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810888397.0A Pending CN108977381A (en) | 2018-08-07 | 2018-08-07 | Raw bacillus amyloliquefaciens and its application in a kind of banana |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108977381A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110317747A (en) * | 2019-06-04 | 2019-10-11 | 华南农业大学 | A kind of bacillus amyloliquefaciens JT68 and its application in prevention and treatment tea anthracnose |
| CN115197869A (en) * | 2022-05-19 | 2022-10-18 | 广东海洋大学 | Bacillus amyloliquefaciens ZJLMBA1908 with broad-spectrum antibacterial activity and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1637133A (en) * | 2004-12-09 | 2005-07-13 | 中山大学 | Banana endophyte and its use |
| WO2011032329A1 (en) * | 2009-09-18 | 2011-03-24 | 南京农业大学 | Antagonistic bacteria for preventing and treating panama wilt disease of continuously planted banana and microorganism organic fertilizer thereof |
| CN102172249A (en) * | 2011-03-24 | 2011-09-07 | 南京农业大学 | Marine bacteria 2BS12 preventing and controlling banana fusarium wilt |
| CN103004886A (en) * | 2012-12-11 | 2013-04-03 | 广东海洋大学 | Marine bacterium preparation for preventing banana vascular wilt |
| CN106676049A (en) * | 2017-03-03 | 2017-05-17 | 广西壮族自治区农业科学院生物技术研究所 | Bacillus amyloliquefaciens strain and application thereof |
-
2018
- 2018-08-07 CN CN201810888397.0A patent/CN108977381A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1637133A (en) * | 2004-12-09 | 2005-07-13 | 中山大学 | Banana endophyte and its use |
| WO2011032329A1 (en) * | 2009-09-18 | 2011-03-24 | 南京农业大学 | Antagonistic bacteria for preventing and treating panama wilt disease of continuously planted banana and microorganism organic fertilizer thereof |
| CN102172249A (en) * | 2011-03-24 | 2011-09-07 | 南京农业大学 | Marine bacteria 2BS12 preventing and controlling banana fusarium wilt |
| CN103004886A (en) * | 2012-12-11 | 2013-04-03 | 广东海洋大学 | Marine bacterium preparation for preventing banana vascular wilt |
| CN106676049A (en) * | 2017-03-03 | 2017-05-17 | 广西壮族自治区农业科学院生物技术研究所 | Bacillus amyloliquefaciens strain and application thereof |
Non-Patent Citations (7)
| Title |
|---|
| JUN YUAN ET AL.: "Antifungal Activity of Bacillus amyloliquefaciens NJN-6 Volatile Compounds against Fusarium oxysporum f. sp. cubense", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| 周维等: "香蕉内生细菌G9R-3分离鉴定及其拮抗香蕉枯萎病菌活性评价", 《西南农业学报》 * |
| 桑建伟: "香蕉枯萎病拮抗内生芽孢杆菌BEB17的分离鉴定及生防潜力分析", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 桑建伟等: "内生解淀粉芽孢杆菌BEB17脂肽类和聚酮类化合物的抑菌活性分析", 《植物病理学报》 * |
| 桑建伟等: "拮抗内生细菌BEB17对4个香蕉品种组培杯苗的定殖、促生分析和抗病性评价", 《热带作物学报》 * |
| 田丹丹等: "香蕉枯萎病菌拮抗内生细菌GKT08鉴定及抗病促生效果评价", 《中国南方果树》 * |
| 陈奕鹏等: "拮抗内生芽孢杆菌BEB17分离鉴定及其发挥性物质抑菌活性分析", 《植物病理学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110317747A (en) * | 2019-06-04 | 2019-10-11 | 华南农业大学 | A kind of bacillus amyloliquefaciens JT68 and its application in prevention and treatment tea anthracnose |
| CN115197869A (en) * | 2022-05-19 | 2022-10-18 | 广东海洋大学 | Bacillus amyloliquefaciens ZJLMBA1908 with broad-spectrum antibacterial activity and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105132336B (en) | One bacillus amyloliquefaciens and its microbial inoculum and application | |
| CN103087951B (en) | Bacillus amyloliquefaciens and application thereof | |
| CN105296381B (en) | One bacillus subtilis CYY-25 and its application | |
| CN106916764B (en) | One plant of acid proof South Korea pseudomonad CLP-7 and its application | |
| CN105132324B (en) | One plant of wheat rhizosphere Paenibacillus polymyxa WXD 6-4 and its application | |
| CN104630121B (en) | Bacillus amyloliquefaciens plant subspecies ZFH-3 microbial bacterial agents and its application | |
| CN102747020B (en) | Bacillus subtilis and application thereof | |
| CN101851597B (en) | Streptomyces griseoflavus for resisting alfalfa diseases and screening method thereof | |
| CN103160442B (en) | Paecilomyceslilacinus strain having strong pathogenicity for diaphorina citri | |
| CN105586298A (en) | Bacillus subtilis, bactericide, preparing method of bactericide and application of bactericide | |
| CN102367421B (en) | Penicilliumgriseofulvum XJ-EP-058 strain as well as microbial preparation resisting banana vascular wilt and application thereof | |
| CN105734000A (en) | Paenibacillus polymyxa NSY50 with capabilities of promoting growth and preventing diseases | |
| CN101760438A (en) | Broad-spectrum antifungal plant endophytic bacillus subtillis and application thereof | |
| CN103114064A (en) | Marine actinomycete with antibacterial activity to multiple plant pathogens | |
| CN108570433A (en) | One plant of acid proof defence pseudomonad CLP-6 and its application | |
| CN102925387B (en) | Bacillus simplex for inducing soybean to generate soybean cyst nematode resistance and application | |
| CN104342388B (en) | Streptomycete bacterial strain and prevent and treat tomato wilt use in conjunction | |
| CN103436465B (en) | Alcaligenes faecalis strain | |
| CN105567600A (en) | Pathogen verticillium antagonistic bacterium and application thereof | |
| CN105441331A (en) | Myrothecium roridum and application thereof | |
| CN108384734A (en) | A kind of biocontrol agent and preparation method thereof of prevention tobacco rhizome class disease | |
| CN103146609B (en) | Pseudomonas fluorescens and method for preventing phytophthora capsici thereby | |
| CN108977381A (en) | Raw bacillus amyloliquefaciens and its application in a kind of banana | |
| CN113604376B (en) | Sugarcane endophytic bacillus subtilis and application thereof | |
| CN103087956B (en) | Bacterium for preventing and controlling banana wilt and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20181211 |